JPH05502369A - Method for producing in vitro proliferating lymphoid cells for use in adoptive immunotherapy - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 31. In vitro proliferated lymphoid cells produced by the method according to claim 6.

32, in vitro proliferating lymphoid cells produced by the method according to claim 14 .

33. Tumor-infiltrating lymphocytes produced by the method according to claim 16.

34. Intestinal tumor-infiltrating lymphocytes produced by the method according to claim 19.

35. Tumor-infiltrating lymphocytes produced by the method according to claim 24.

36. Hollow fiber extraspace cell supernatant produced by the method according to claim 12.

37. A hollow fiber extraspace conditioned medium produced by the method according to claim 13.

38. Hollow fiber extraspace cell supernatant produced by the method according to claim 22.

39. A hollow fiber extraspace conditioned medium produced by the method according to claim 23.

40° An improved method for producing in vitro proliferating lymphoid cells comprising an effective amount of hollow culturing the cells in the presence of an extrathalamic space-conditioned medium, wherein the chain members Stimulating the growth rate of proliferating cells so that they grow less than they would in their absence. How to increase it by at least about 50%.

Specification used in adoptive immunotherapy Method for producing in vitro proliferating lymphoid cells This invention is disclosed in U.S. Patent Application No. 07/238°445 filed August 31, 1988. are continuation-in-part applications, the subject matter of which is in the therapeutic treatment of diseases to which they refer. A very promising recent development is the use of adoptive immunization (e.g. υrologic Oncology, edited by Lepor et al., Kluwer Academic Publishers, Boston, Chapter 12, Belldegrul et al. reference). Adoptive immunization involves the in vitro proliferation of lymphoid cells (in It is a passive transfer of vitro expanded lymphoid cell. . The in vitro expanded lymphoid cells are therapeutically effective in treating disease. this is, Destroys affected host cells by interactions specific to the affected host cells. by destroying or by giving the host a therapeutically effective substance. be.

Adoptive immunization involves the isolation of lymphoid cells from an affected individual, the isolation of lymphoid cell subpopulations (su bpopl,ll! selective expansion of tion) and large numbers (1× 101O to 5X10”), thereby allowing in vitro expansion. Lymphocytic cells destroy cancerous or other affected cells or are therapeutically effective. supply substances; The lymphoid subpopulation is responsible for cancerous or other affected cells. have a high degree of reactivity, or are capable of expanding subpopulations of cell populations in vitro. In the presence of growth-promoting substances such as interleukins that mediate Modified to have reactivity. Growth stimulation for some adoptive immunotherapies In addition to the stimulating substance, lymphoid cells increase in the presence of the target antigen or cells representing the target antigen. be cultivated. Cells displaying the target antigen are generally derived from an affected individual. The target antigen is can be isolated from the cells of an affected individual or by synthetic means, e.g. recombinant They can be prepared by DNA technology or peptide synthesis. Nutrition for cancer treatment The use of offspring immunity has been widely studied (e.g., Belldegrun et al., cited above). (see). Adoptive immunization can be used to treat cancers of the kidney, colon, lung, kidney or chest, melanoma, lymphoma, etc. It is used for the treatment of tumors and endometrium (for example, the aforementioned Bet ldegru n et al., Tapalian et al. (1988) 1. Cl1n, 0nco! , 5 :839-853, Rosenberg (1987a) U.S. Patent 4.69 No. 0.915, the disclosures of which are hereby incorporated by reference in their entirety. Incorporated; others, Rosenberg et al. (1987b) NewE ng, l, Med, 316: 889-897, Il! lportamt Advances InOncolog7. Edited by DeVita et al., IB Lip Rosenberg, New York, pp. 55-91. reference).

Adoptive immunization is also used to treat other diseases, including viral diseases and genetic defects. You can also be there. For example, isolating lymphoid cells from patients and correcting gene defects or to code for a therapeutically effective agent. Therefore, by incorporating cloned DNA into the DNA of these lymphoid cells, changes can be made. be incubated, cultured, and reintroduced into a patient in which the cloned DNA is expressed. Can be done. Presence of specific viral target antigens and specific factors such as IL-2 Immunoreactive lymphoid cells cultured under may be useful for therapy or immunization.

As mentioned above, lymphoid cells are exposed to viral antigens or immunologically active viruses. In the presence of a specific antigen, such as a natural protein or an antigenic site of an antigen, or cells containing such antigens, such as tumor cells or virus-infected cells. When cultured in the presence of immunocytochemistry, some adoptive immunotherapies It requires the use of a proliferating population of lymphoid cells generated from paralytic cells. used The antigen may be present on the cell surface, such as on irradiated tumor cells, or purified antigen may be present on the cell surface, such as on irradiated tumor cells. or expression of cloned DNA or peptide synthesis. It may also be a synthetic antigen produced by a method. The source of the antigen is tumor cells, When tumor cells are cultured in the presence of IL-2, the cells they produce are more likely to invade the tumor. They are lymphocytes (hereinafter referred to as TIL).

The necessary inclusions for the proliferation of such tumor-specific lymphoid cells are growth-promoting substances. , mitogens, cytokines and lymphokines. Mitogen is In response to antigen-independent growth of lymphoid cells, cytokines influence other cells. Lymphokine- or monokine-like factors produced by human cells. Lymphokines are produced and secreted by activated T lymphocytes and have effects on other cell types. It is a substance that affects

In particular, certain lymphokines, such as IL-2, have specific surface phenotypic markers. and a subpopulation of lymphoid cells that specifically recognize specific antigens on the surface of affected cells. It is known to mediate specific proliferation. Interleiki, a lymphokine IL-2 (hereinafter referred to as IL-2) is a specific lymphocyte population that has high antitumor activity. used for propagation (e.g. Rosenberg (1987a) mentioned above). reference. In addition, the aforementioned Rosenberg (1987b), Rosenbe rg (1986a), the aforementioned Yron et al., the aforementioned Rosenberg (1986a), the aforementioned Yron et al. 985)).

IL-2, originally identified as a T-cell growth factor, can be expressed in either isogenic or allogeneic forms. Specific lymphoid cells with antitumor reactivity against hosts affected by genetic tumors It is used to generate. For example, from tumor-affected hosts including humans and mice. The resting lymphocytes obtained were incubated in the presence of IL-2 for 3 to 4 days. It is possible to lyse natural killer cell (NK)-resistant tumor cells. However, normal cells result in proliferation of lymphocyte plate populations that do not lyse (e.g. See, eg, Belldegrun et al., supra). This phenomenon is caused by lymphokine activation This phenomenon is called LAK killing, and there are two types of lymphocytes that respond to this phenomenon. Consists of cells. The first type of cells are called LAK cells, and the second type of cells are called LAK cells. is called TIL.

LAK cells can be found in both normal individuals and individuals suffering from cancer or other diseases. can be obtained from LAK cells are composed of NK and cytolytic TIJ lymphocytes. They appear to constitute a cell lysis system distinct from CTL cells (bottom). L.A.K. Cell precursors and effector cells have phenotypes typical of NK cells (e.g. For example, Phllips et al. (1986) J, EKL Med.

164:814) is a fresh, uncultured NK-resistant allogeneic origin or metastasis. It has the ability to lyse cancer cells, and has the ability to lyse peripheral blood lymphocytes (PBL), thymus, spleen, It can be generated from lymph nodes, bone marrow, and thoracic duct cells (e.g., as previously described). Bellegrun et al., pp. 215-216). In addition, LAK cells lysed fresh autologous and allogeneic tumor cells, as well as many cultured cell lines. have the ability to understand LAK cell precursors have both T and B cell surface markers. LAK cells are Thy1.2-positive and Ia-negative, and most of the cytotoxic activity appears to be in the FcR-positive subpopulation. (For example, Functions of the Natural Immun eSystemS Reynolds et al., Plenum Pub, Corp. (See Lefor et al. (1989), pages 39-56). In addition, LAK cells has been shown to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) ( See, e.g., Lefor et al., supra). IL-1 and tumor necrosis factor (TNF) enhanced such IL-2-induced ADCC activity, and such enhancement was induced. It has been shown that Ei Senjhal et al. (1989) J, of Iamunol, 142: 2307-2313).

TIL cells infiltrate into tumors where the immunological response of the host's immune system is heightened. lymphocytes, from which they can be isolated (e.g., Yron et al. et al., and ^nder@on et al., supra). TIL cells are autologous cells It has higher specificity than LAK cells for cancer adoptive immunotherapy. have been found to be more potent than AK cells (e.g., see Yton et al., supra). (see). TIL cells fight kidney, colon, and breast cancer, melanoma, and sarcoma. Obtained from resected human tumors, including:

Cells obtained from tumors and grown in the presence of IL-2 were incubated in vitro. By activating active T cells within the tumor and tumor cells or tissue destruction occurs. After 2-3 weeks of culture, all tumors were destroyed. The culture consists of lymphoid cells with a cytolytic T lymphocyte (CTL) phenotype. (e.g. Muul et al. (1987a) J, 1mmunot 138: 98 9, the aforementioned 7opaliao et al. and Hoh et al. (1986) Cancer Res, 46:3011). Some human TIL cells have their Shows high specificity for autologous tumors.

TIL cells also show promise for use in gene therapy methods (e.g. Cu 1liton (1989), 5eienc! 244: 1430-143 (See “News and Commet” in Section 3). They are the desired tamper denatured by inserting DNA encoding the protein, cultured, and reintroduced into the patient. Provides a source of autologous cells that can be introduced. Desired protein for cancer treatment tumor necrosis factor, the CD4 receptor that HIV binds to, and the host where the treatment is administered. It may be a therapeutically effective protein, such as an enzyme that is missing or a treatment markers that allow studying the fate of TIL cells in hosts subjected to It may be a protein.

In addition to LAK and TIL cells, other lymphoid cell types also exhibit antitumor reactivity. It has been found that For example, RLN (regional draining lymph node (+egi) anal draining lymph node) cells exhibit antitumor activity. derived from tumor-bearing mice immunized with weakly immunogenic tumors (Slephenson et al. (1989) Surger7105: 52 3-528), RLN cells are therapeutic effector cells, and LAK cells indicates a different cell population.

Because many cancer patients do not respond to adoptive immunotherapy, alone or in combination with IL-2 for the expansion of lymphoid cell subpopulations to Identify other lymphokines, cytokines and/or mitogens that may be useful in Research is underway. IL was first used to generate lymphoid cell subpopulations. -2, but other lymphoids such as IL-4, IL-6 and interferon Hokine, and TNF are also useful for producing expanded lymphoid cells in vitro. has been shown to be useful for the proliferation of specific subpopulations of lymphoid cells. It will be. For example, IL-4 (also called BSF-1) is derived from T cells. It is a glycoprotein that activates LAK if lymphoid cells are initially stimulated with IL-2. but has been shown to be suppressed if the cells have not been stimulated beforehand. (Xawakami et al. (1989) J, ol. ImmunoL 142 2 3452-3461).

IL-4, alone or together with IL-2, also stimulates TIL cell growth. It has been shown that it can be irritating. IL-4 enhances TIL cell growth and Concomitantly, it seems to inhibit the growth of NKHf cells that respond to non-specific killer activity. It looks like (Hunun Tumor Antigens and 5pCcif ic Tumor Thelip7^tan R, Li5s, Inc. See Lotze et al. (1989), pp. 167 to 179, and others, ka wakami et al. (1988) 1. ofExptal, Med, 168: 2183-2181).

The possibilities for adoptive immunotherapy are almost limitless. Specificity between lymphoid cells and tumors Not only can it be used in the treatment of cancer due to the interaction of , can be used to treat genetic diseases, and can be used to deliver anti-tumor and other therapeutic agents. It can also be used as a stage. Lymphoid cells produce enzymes or hormones It is possible to eliminate it from individuals suffering from genetic diseases that result from the defect. interest A wild-type copy of the gene targeted by the lymphocyte can be inserted into the lymphocyte, and this Paracytes can be cultured in the presence of selective agents and reintroduced into afflicted individuals.

Alternatively, a therapeutically effective anti-inflammatory drug such as interferon or TNF may be used. Lymphoid cells are genetically modified to express cancer drugs or antiviral drugs to treat patients. It can also be reintroduced to humans (eg, Genejic l!ng.

News, vol. 9, Noj, March 1989, and Businesa Week/May 1, 1989, p. 133).

In the implementation of adoptive immunotherapy, methods for identifying therapeutically useful lymphoid cell subpopulations are being developed. We need a way to not only generate but also produce large numbers of such cells. human Although adoptive immunotherapy for cancer and other diseases is a very promising method, it has not been clinically effective. Adoptive immunization is characterized by the inability to generate adequate numbers of immunoreactive lymphoid cells. are major barriers to accessing therapy (e.g., Rosenberg et al. (1986b) 5science 233:131g-1321, and previous (see Culliton, supra). For this reason, methods for large-scale production of lymphoid cells and can be easily adapted if new cells or agents for growth and proliferation are identified. There is a need for a method to

Currently, there are enough drugs such as TTL to be administered to patients with metastatic disease. Generation of proliferating populations of lymphoid cells is time consuming, inefficient, and prohibitive It is expensive.

This allows TILs derived from metastatic lesions to be purified using human serum albumin and recombinant protein, respectively. Plasticontaining approximately 1.5 liters of tissue culture medium containing human IL-2. This is achieved by growing in gas-permeable culture bags made from Muff et al. (1987b) J. ImmunollMeth, 101: 171-181; see also Cullijon, supra). cells are the most Grow and divide until a maximum density of 2-3X 109 cells per bag is reached. Ru. At maximum concentration they must be divided into additional culture bags. others To generate a sufficient number of cells for a single patient-to-person clinical procedure, approximately 50 Therefore, 150 bags are required. until enough cells are generated for clinical treatment. The cells are grown for 4 to 6 weeks and divided into additional bags, typically are grown into 100 to 150 bags.

This bag contains approximately 1011 cells. After that, 151) to 250 Cells in a liter volume must be centrifuged and maintained under sterile conditions. Alone The entire process of generating enough cells to treat patients takes a lot of time and manpower. In addition, large amounts of mammalian cell culture media, human serum albumin and IL-2 are used. I can stay. Typically, approximately 5 hours are required to harvest a single treatment dose of cells. . This cell concentration method produces cells in dilute solution, has many opportunities for cultured cell contamination, and is prohibitively expensive. Therefore, its use as a means of generating therapeutically necessary amounts of cells for clinical procedures is extremely difficult. limited to.

Therefore, a large number of biologically active organisms are suitable for use in adoptive immunotherapy. Those who can utilize the efficient and cost-effective growth of exocytic lymphoid cells. Laws need to be developed. Furthermore, a wide range of diseases can be treated by this method. Modified protocols and proliferating parallel populations of lymphoid cells are utilized to cover a wide range of There is a need to develop methods that can be easily adapted to the many protocols that exist.

Summary of the invention One of the objects of this invention is to provide in vitro expanded lymphocytes that can be used for adoptive immunotherapy. An improved method for large scale production of sex cells comprising: (a) hollow fibers of a hollow fiber bioreactor; A suspension of lymphoid cells in a growth-promoting factor-containing medium was inoculated into the external space, and b)! A growth-promoting substance that specifically proliferates a therapeutically useful subpopulation of J-pathogenic cells. Perfusing the bioreactor with tissue culture medium containing an effective amount of at least one Here, the effective amount is an amount sufficient to cause specific growth, and the tissue culture medium is maintain cell division and growth of subpopulations, and the therapeutic use of adoptive immunotherapy and (C) placing the cells in the bioreactor in the presence of a growth promoting substance. culture for a sufficient period of time to obtain a therapeutically effective number of proliferating lymphoid cells in vitro. An object of the present invention is to provide a method for providing the following.

Another object of this invention is to produce in vitro proliferating lymphoid cells for use in adoptive immunotherapy. an improved method for producing cells, wherein the proliferating lymphoid cells are TIL cells; (b) suspending cells from resected tumor tissue in tissue culture medium; (b) adding the suspension to the tumor-infiltrating region an effective amount of at least one cytokine capable of promoting lymphocyte proliferation; cultured in the presence of tumor-infiltrating lymphocytes in suspension, wherein the effective amount (c) is a component of the hollow fiber culture system; A cultured suspension of tumor-infiltrating lymphocytes is attached to the outer space of the hollow fiber bioreactor. and (d) at least one species capable of promoting the proliferation of tumor-infiltrating lymphocytes. Perfuse the bioreactor with tissue culture medium containing an effective amount of cytokines. Here, the effective amount is an amount sufficient to cause specific proliferation, and the effective amount is an amount sufficient to cause specific proliferation, and The medium maintains cell division and growth of tumor-infiltrating lymphocytes, and (e) tumor-infiltrating Therapeutically effective lymphocytes are harvested in the presence of tissue culture medium in a bioreactor. The present invention provides a method comprising culturing for a sufficient period of time to obtain a sufficient number of tumor-infiltrating lymphocytes. It's about doing.

Another object of this invention is to provide in vitro expanded lymphoid cells for use in adoptive immunotherapy. It is about providing.

Another object of this invention is to provide in vitro expanded lymphoid cells for use in adoptive immunotherapy. wherein the proliferating lymphoid cells are TIL cells.

Another object of this invention is to provide in vitro expanded lymphoid cells for use in adoptive immunotherapy. wherein the proliferating lymphoid cells are provided with cloned DNA. These are lymphocytes whose genomes have been degenerated due to

Another object of this invention is to stimulate the growth of proliferating lymphoid cells in vitro and to A biologically active agent that specifically proliferates lymphoid cells in vitro and is useful for therapeutic purposes. A method for producing a conditioned medium used as a source of growth promoting substances, the method comprising: Remove the contents of the extra-hollow fiber space of the bioreactor where lymphatic cells are cultured. , pelleting and removing cells from the contents of the hollow fiber outer space; generate intercellular cell supernatant and transfer the hollow fiber extracellular cell supernatant to tissue culture medium. An object of the present invention is to provide a method comprising preparing a hollow fiber extraspace-conditioned medium by dialysis. Ru.

Another object of the invention is to stimulate the growth of proliferating lymphoid cells in vitro, and A biologically active compound that specifically proliferates therapeutically useful in vitro proliferating lymphoid cells. The object of the present invention is to produce a conditioned medium that can be used as a source of growth promoting substances.

Another object of the invention is an improved method for producing expanded lymphoid cells in vitro. and culturing the cells in the presence of a dynamic hollow fiber extraspace conditioned medium. and said amount stimulates the growth rate of the proliferating cells, thereby inhibiting the growth of the cells in their absence. An object of the present invention is to provide a method of increasing the amount of the compound by at least about 50%. Ru.

This invention provides an in vitro expanded lymphoid cell system that can be adapted to the specific requirements of adoptive immunotherapy procedures. By providing an improved method for culturing cells, therapeutically useful amounts of proliferating lymphoid cells can be obtained in vitro. greatly improves the procedure for preparing cells. In this improved culture method, lymphoid cells is obtained from a patient, inoculated into a hollow fiber bioreactor culture system, and treated At least one type of growth promoter that specifically proliferates a therapeutically useful lymphoid cell subpopulation cultured in the presence of an effective amount of the substance.

By practicing this invention, biologically active therapeutic agents can be obtained using any convenient method. A therapeutically useful yield of in vitro expanded lymphoid cells is obtained. This is before This not only significantly reduces the cost of manufacturing cells, but also increases the number of cells that can be manufactured. significantly increase the number.

Brief description of the drawing FIG. 1 represents a typical growth curve for TIL growth according to the method of this invention. TIL The number of cells has increased over time since the date of excisional biopsy. cells are patient Obtained from tumor excised from W. Enzyme-dispersed in standard culture flasks, in a gas permeable bag when the TILs have grown sufficiently from the TIL-containing tumor. Cultures were started and then inoculated into hollow fiber reactors 23 days after the date of excisional biopsy. . -〇-0-0- indicates the growth curve of cells grown in gas permeable bags. vinegar.

This is the incubation period plotted against the abscissa in days versus the ordinate on the right. It is evaluated by the increase in the number of cells plotted as After an initial lag period, patient W The number of cells obtained (W-TiL) increased exponentially during the period. Time = O Indicates the date of excisional biopsy. TIL cells were pulled from one bag on day 23 and cultured in C. E L LMAX”I’1lEl empty fiber bioreactor was inoculated and rotated on the 44th day. I got it (-moo-moo-). Glucose consumption increased logarithmically with time ( −〇−〇−0−＞.

This is the concentration of particles in the bioreactor perfusate, plotted against the ordinate on the left. It is evaluated by the decrease in lucose concentration versus the number of days of culture on the abscissa. + is hollow fiber outside Replacement with fresh complete medium during (EFS) is shown. 4.3X to the hollow fiber on the 23rd day By inoculating 10" cells, 5.4X 1010 cells will be collected at the time of collection. Obtained.

Figure 2 shows the number of G-TILs (patient G FIG. 2 shows a growth curve showing the TIL obtained by -〇-0-0- is gas permeability A growth curve is shown showing the increase in cell number for cells grown in the bag. early lag stage Afterwards, the number of cells obtained from patient G increased exponentially during the measurement period from the day of excisional biopsy. added. On the 16th day, I pulled out TIL from one bag and it was in CELLMAXTM. Empty fiber bioreactors were inoculated and harvested on day 30 (-moo-moo-moo). gel Call consumption increased logarithmically with time. This is the pro- gram for the left ordinate. Decreased glucose concentration in the bioreactor perfusate versus culture period It has been evaluated as follows. + indicates replacement of the extra-hollow fiber space (EFS) with fresh complete medium. vinegar.

By inoculating 1.0X10 TILs, 1.5X 1010 TIs L was recovered. Perfusion of the hollow fiber bioreactor was performed on the first collection date of 30 Reset on day 51, and any remaining TILs will be allowed to grow again at the second harvest on day 51. Ten 1.5XlO pieces were collected. Reset the perfusion again and repopulate the remaining cells. At the third collection on day 73 of breeding, 2. 1×1010 cells were collected.

Figure 3 shows this inside the hollow fiber space of the “CELLMAX” hollow fiber bioreactor. Figure 1 shows a scanning electron micrograph of TIL cells from patient W grown by the method of the invention. vinegar.

In Fig. 3a, the space between the giant egg-shaped hollow fibers is filled with solid lump-like TIL. It is being The valve-shaped shape of the normally cylindrical hollow fiber and the space between the TIL mass and the hollow fiber surface The space is an artifact of histological adjustment.

Figure 3b shows such a structure that it is possible to see individual cells near the outer surface of a single hollow fiber. It shows a higher magnification.

Description of preferred embodiments Unless otherwise defined, technical and scientific terms used herein refer to has the meaning commonly understood by one of ordinary skill in the art. All publications mentioned here are , incorporated herein by reference.

As used here, lymphoid cells refer to the presence or absence of lymphoid cells. Also included are lymphoid cells derived from other tissues.

Generally, lymphoid cells are removed from the individual to be treated. lymphoid cells tumor, peripheral blood, or lymph nodes that harbor or produce lymphoid cells and It may also be derived from other tissues such as spleen.

As used herein, adoptive immunotherapy thereby The process of transporting, administering, or introducing cells into an individual or host.

When lymphoid cells are cultured in vitro under appropriate conditions, their characteristics Certain subpopulations selectively proliferate. The generated cell proliferation population is here described as a living organism. They are called exoproliferating lymphoid cells. This subpopulation of cells generally has distinct phenotypes It is a heterogeneous mixture of cells, but it can also consist of a homogeneous population of cells. Generate A particular mixture of cells is produced by the production of starting materials and conditions to produce such cells. It is for fune+in. Proliferating populations can be used in adoptive immunotherapy protocols. I can do it. By introducing it into the individual to be treated, a proliferating population of cells is induced. , which specifically recognizes and in some sense mediates the destruction of the affected cells of the host, and/or produce a therapeutically effective agent. Conditions under which such cells generate IL-2,! Cytokines such as L-1, IL-6 and IL-4 may growth in the presence of a mixture of cytokines. Lymphoid cells are supported When cultured in the presence of cytokines, the lymphoid cells produced are The in vitro proliferating population includes activated lymphoid cells and their sources and uses. Includes LAK cells and TIL cells, depending on the cytokine involved There is. Lymphoid cells that proliferate in the presence of cytokines are tumor-derived In some cases, the proliferating lymphoid subpopulation of cells that result is referred to as TIL cells. Lymphoid When cells are grown in the presence of cytokines and target antigens, the These cells are called activated lymphoid cells here. the target antigen is present on the cancerous cell; or, if derived from them, in vitro proliferation of activated lymphoid cells. A subpopulation is TIL cells.

As used herein, a therapeutically useful subpopulation of in vitro expanded lymphoid cells is This is a subpopulation that can be used for adoptive immunotherapy.

As used herein, in vitro expanded lymphoid cells are used for adoptive immunotherapy. cells that are cultured for Such cells include at least one lymphoid cell. lymphoid cells in the presence of specific inclusions that induce proliferation of specific populations of species. It corresponds to a subpopulation of lymphoid cells that is produced when cultured.

Such subpopulations include those that have immunological reactivity with the affected cells of the affected patient. Includes: If such cells are used as therapeutic agents, they will be discussed here. These are called immunologically reactive lymphoid cells. When used in adoptive immunotherapy for cancer Examples of immunologically reactive cells are TIL and LAK cells. In vitro propagation Other subpopulations of lymphoid cells have been genetically engineered to Lymphoid cells that have been modified to contain DNA encoding proteins that cannot be produced included. Examples of such proteins include traceable foreign marker proteins. , and therapeutically effective substances such as CD4 to which HIV binds and anti-cancer drugs. It will be done. In addition, such cells can be selectively expanded or destroyed in vivo. Genetic DNA that encodes drug resistance or drug sensitivity may be processed. Other subpopulations have specific target antigens in vitro. Lymphoid cells cultured in the presence of. Target antigen (alone or plural) may be present on the surface of cells, such as tumor cells, from cells, or It may also be prepared synthetically and introduced into tissue culture media. The target antigen is a peptide compound. It may be synthesized by a synthetic method or by genetic engineering. In general, living organisms Exoproliferative lymphoid cells are cultured in the presence of the target antigen and are infected with the target antigen. specifically recognize and in some sense mediate in the destruction of target cells or attack target antigens. This is the cell that delivers the therapeutic agent to the treated cells.

As used herein, the target antigen is a subpopulation of lymphoid cells proliferating in vitro. It is an antigen that is specifically recognized.

An effective amount for at least one target antigen is an amount for an organism that specifically recognizes this antigen. The amount is sufficient to select at least one subpopulation of exproliferating lymphocytes.

As used herein, tumor-specific in vitro proliferating lymphoid cells are Alternatively, it is a cell that specifically recognizes a target antigen present within tumor cells. TIL The cells are tumor-specific lymphoid cells. As used herein, the tumor-specific antigen is , an antigen present on or within tumor cells. Tumor-specific antigens Can be used in pure form on tumor cells or by purification from tumor cells. or synthesized in vitro by methods such as genetic engineering. can be obtained.

When used in two cells, the growth-promoting substance stimulates cell growth and/or division. It is a substance that participates or induces in a certain sense. Growth-promoting substances include mitodi This includes enzymes and cytokines. Examples of growth promoting substances include fibroblast growth factors. children, epidermal growth factor, oncogene products, interleukins, colony stimulating factor, and other factors known to those skilled in the art.

As used herein, mitogen is a substance that induces cell division, especially , as used herein, stimulates and grows lymphoid populations in an antigen-dependent manner, It is a substance that causes differentiation into effector cells.

As used herein, cytokines are produced by cells that affect other cells. agents such as lymphokines or monokines.

As used herein, lymphokines are produced and secreted by T lymphocytes. , are substances that affect other types of cells. Tumor necrosis factor, interleukin Interferon and interferon are examples of lymphokines. Monokine is a monocyte or is a substance secreted by macrophages that affects other cells.

As used herein, a therapeutically effective number of in vitro proliferating lymphoid cells refers to at least sufficient to achieve the desired therapeutic effect when used in standard adoptive immunotherapy. is the number of cells.

For example, a therapeutically effective amount of T1L for a single treatment of a patient suffering from metastatic cancer. is at least about 10 to 10 cells.

As used herein, a hollow fiber culture system allows the medium to pass through the system. pumping means to perfuse the medium, storage means to supply and collect the medium, and electricity. from the hollow fiber bioreactor, in addition to other elements including control, recording and sensing equipment. Become. A hollow fiber pi reactor consists of a large number of semi-permeable tubes enclosed within a hollow shell. It is a cartridge made of shaped fibers. Hollow fiber reactor and hollow fiber bis The term reactor is used interchangeably.

As used herein, extra-hollow fiber space (EFS) is the space in which cells grow. within the shell of the hollow fiber bioreactor, and comprising semipermeable fibers. It is the external space of

As used herein, EFS cell supernatant is defined as EFS cell supernatant in which cells within the EFS are growing. It is a medium. This includes secreted cellular products, diffusible nutrients, and Lymphokines and services that mediate the specific proliferation of proliferating lymphoid cell subpopulations in vitro. Contains growth-promoting substances such as itokine.

As used herein, the EFSH conditioned medium is centrifuged to remove cells and certain substances. EFS cell supernatant after removal of and dialysis against tissue culture medium.

As used herein, the selective medium refers to the culture produced by lymphoid cells proliferating in vitro. a tissue culture medium containing the desired growth promoting substance(s) and other selections; cells that are selected to grow with an agent, e.g., a target antigen or gene treatment. It is an active substance.

As used herein, Complete AIMV refers to the proprietary formula AI Consists of M-V (GIBCO5Grand l5land, N, Y,) , and further 1000 units/m 1 1 L-2 (manufactured by Cejus, Eme ryville, CA, ), 10 μg/m 1 dientamicin (GIB CO), 50μg/ml streptomycin (GIBCO), 1.25ag /ml fBizone CFIow Laboralo rles.

MacLeaa, VA, ), 2.95mg/rn 1gluco-branch and and 2mM glutamine (Flov Laboratories). It is a medium. Supplemented complete AI M-V medium is obtained from autologous LAK cell cultures. consisting of complete AIM-V supplemented with 20% AIM-V supernatant.

As used herein, the AIM-V supernatant is as described by Muul et al. (1986) 1. Prepared as described in 1mmuno11Methods 88:265 Ru. Briefly, LAK AIM-V supernatant transforms peripheral blood lymphocytes into AI -V or other suitable tissue culture medium in the presence of IL-2 for 2 to 3 days. Prepared by growing and removing cells by centrifugation to obtain supernatant.

Other suitable tissue culture media are known to those skilled in the art, are readily available, and are It can be easily replaced with AIMV. For example, 10% heat inactivated human a medium consisting of a 50-50 mixture of complete AIM-V and RPMI containing serum; and a medium further supplemented with LAK supernatant can be used.

In carrying out the embodiments of the invention disclosed herein, as a first step, a desired living organism is Lymphoid cells that can be used to generate an exproliferating lymphoid cell subpopulation are generally must be removed from the diseased individual to be treated using adoptive immunotherapy. No. Lymphoid cells include PBL, lymph nodes, lung cells and immune response. It is present in many tissues of the body, including those in the body. Expected to be used in this invention lymphoid cells whose differentiation and growth make them suitable for adoptive immunotherapy in vitro. which can be controlled by appropriate growth-promoting substances to produce lymphoid cells. be.

Cells obtained from the patient are suspended in a cell culture medium suitable for maintaining mammalian cell growth. make it muddy Cell suspensions can be obtained from tumors or other affected tissues, or from lymphoid It can also be prepared from cells. Such media are readily available and , the selection of an appropriate medium is within the level of skill in the art. Then the desired exemption It also contains the growth-promoting substances necessary for selective expansion of the epidemic-reactive lymphocyte plate population. For further growth in a selective medium, cells are placed in culture plates or as described in the art. or by other suitable means known to the person concerned. This medium may contain the target antigen. Thin Cultivate the cells and increase their numbers until sufficient numbers are obtained. Often at this point, The cells are inoculated into gas permeable bags and treated with the desired growth promoter and other desired selective agents. Grow in selective medium containing until at least about 108 cells are produced. . Because cells are cultured in selective media, cells in suspension are primarily of interest. Includes subpopulations. The generated cells are injected into a hollow fiber bioreactor and cultured. The intention is to expand those subpopulations that can be used for child immunotherapy. Culture under appropriate conditions.

Hollow fiber bioreactors (herein referred to as HF) are known to those skilled in the art (e.g. , Xn1zek et al., U.S. Patent No. 4.220.725,4, 206.015,4 ．． See No. 200.689, No. 3.883.393, and No. 3.8211087. , the disclosures of which are incorporated herein by reference). hollow fiber bioli Actors are responsible for the growth of mammalian cells and the biologically active substances secreted by them. (see, e.g., Knuek et al., supra; et al. Yoshida et al., U.S. Pat. No. 4.391.912 HMeyers et al., U.S. Pat. No. 4.546.083; and Markus et al., U.S. Pat. No. 4.301.24. (See No. 9).

The hollow fiber bioreactor to be used in carrying out this invention is It has a large number of tubular semipermeable membranes (hereinafter referred to as fibers) housed in an empty shell. Ru. Cultured cells grow to fill the spaces between the fibers and perfuse through the lumen of the membrane. Diffusion or flow of nutrients from the medium is provided. In carrying out this invention Examples of hollow fiber pyreactors that can be used include hollow fiber bioreactors, B3, Ce1lc.

Advanced Bioorgactors, Inc., KeIlsiBjo n, MD, (a complete description of which can be found in the aforementioned U.S. patent application Ser. No. 07/238.44) 5).

The bioreactor, B3, has approximately 8000 tubular semipermeable membranes, which are 1.1 m Give the surface area of 2. Each fiber is about 250 μm in diameter and has a length of about 12 inches. is pulled through the recarbonate tube and further through the lumen of the fiber. Each end is sealed so that the liquid flows and drains at opposite ends of the shell.

The fiber wall nominally allows the passage of substances with molecular weights below the desired cut-off range. Limit the top. The fibers hold the cartridge in a volume typically around 50ml. Divide into extra-fiber space (EFS) and fiber lumen volumes. Fiber and and the shell form a hollow fiber cartridge. Minimum amount of liquid inside the hollow fiber space Bulk flow occurs. This space can be used as an external capillary or shell-side space. Also called.

If desired, filter selected target antigens and/or growth promoters prior to use. You may bring it to the fiber. Fiber targets antigen and/or growth-promoting The materials must be selected in such a way that they can bind. Bonding is a crosslinking agent or by other methods known to those skilled in the art. or by adsorption of antigens or substances onto the fibers. It may also be reversible.

A hollow fiber bioreactor is one component of a hollow fiber cell culture system. mentioned above Knaxek et al., U.S. patent application Ser. No. 07/238.445, the entire disclosure of which In CELLMAX 1100T, the body is incorporated herein by reference. Empty fiber cell culture system (Cellco Advanced Bioreacto) Typical hollow fibers such as TS, Inc., Kensingio3 MD5) The cell culture system consists of standard glass media bottles, stainless steel, which serve as reservoirs. Steel/Ryton gear pump, fiber and shell casing The cells can be autoclaved (al.

clavable) hollow fiber bioreactor, and the appropriate pH and Medical grade silicone that acts as a gas exchanger to maintain air and pO2 Consisting of rubber tubing or other connection means. All elements are standard system Stainless steel with dimensions small enough to fit within tissue culture incubators Fixed to the tray. Automatic reversal of pump speed and flow direction A flap that is installed outside the incubator and passes through the incubator door gasket. determined by an electronic control unit connected to the pump motor by a rat ribbon. determined. The pump motor is magnetically coupled to the pump and is steam autoclaved (+ team autoclaviB). target antigen and/or a tissue culture medium that may contain growth promoting substances such as IL-2. The soil is siphoned from the reservoir and pumped through the hollow fiber lumen and then into the gas tank. through a gas exchange line where it is reoxygenated and recirculated for subsequent recirculation before returning to the reservoir. and pH readjustment. The flow rate increases as the number of cells increases with time. can be set. Typically, the initial flow rate of the medium is adjusted to about 40 ml/min. Therefore, the time increases to approximately 300 m and 17 minutes.

The direction of perfusion of the medium through the hollow fiber lumen is determined by the nutrient supply within the space surrounding the hollow fiber. periodically and automatically to obtain a more even distribution of cells, discard dilutions, and , typically inverted every 10 minutes.

The entire system is sterilized prior to cell incubation and is designed for Ru. During seeding, cells are placed on the surface of the hollow fiber. Nutrients pass through this hollow fiber. The metabolic waste products enter the interior of the hollow fibers for high-volume recirculating perfusion. Diluted in liquid. Selected fibers allow passage of nutrients into the EFS It should be semi-permeable so that cells can grow on or near its surface. The material should be long-lasting. Fibers are porous and transparent. DEAE-cellulose or polypropylene suitable for mammalian cell growth Made of materials such as For example, if the wall has a nominal molecular weight of less than 3000 Daltons, A 12-inch long cellulosic hollow fiber that restricts the diffusion of substances containing It is suitable for use in carrying out research. In some embodiments of this invention, the Constant growth of lymphoid cells in the presence of antigens recognized by active cells Similarly, tumor cells [7] or target antigens can be reversibly or irreversibly transferred to fibers. is connected to In other embodiments, the growth promoting substance is attached to the fiber. It is. Binding can be reversible, such as by adsorption, or by antigen or If a crosslinking agent is used to permanently bond the growth promoting substance to the fiber It is irreversible. Alternatively, growth promoting substances and/or antigens may be added to the perfusate and /or It can also be included in EFS.

The cell suspension is typically placed within the extra-hollow fiber space (EFS) of the hollow fiber PI reactor. It is inoculated through one of two side ports. The lumen is irrigated with cell culture medium. Flow. This medium contains diffusible nutrients and can be used for adoptive immunotherapy. Contains growth-promoting substances that specifically proliferate subpopulations of lymphocytes Select one or more growth-promoting substances that have good growth potential and also contain target antigens. This is a function of the desired lymphocyte cell subpopulation. This selection is based on the technology in the field. range of levels and may be dictated by the particular lymphocyte cell subpopulation that is desired. It is. For example, if TIL cells are growing, they can be isolated from tumor tissue in several ways. growth and possibly devel of TIL cells. The culture medium contains IL-2, which functions in the management of There should be. Those skilled in the art will be able to determine the growth promoting substances such as Lymphin, which contains IL-2. It is available for purchase. Many such as IL-2 have been cloned and are biologically active. It is expressed in a sexual manner. Growth promoters produced recombinantly, e.g. Preferred for use in the present invention are interleukins produced by stomach. Means for cloning DNA encoding such proteins and methods for cloning such proteins Methods for producing biologically active proteins from digitized DNA are within the skill of the art. It is surrounded. For example, interleukinae-6 has been cloned. many kinds growth-promoting substances and combinations thereof to expand desired lymphocyte cell subpopulations. It may also be used to

In a typical embodiment of the invention, the cells are provided with at least one immunoreactive lymphocyte. in the presence of at least one growth-promoting substance that specifically proliferates a subpopulation of spherical cells. It will be appreciated by those skilled in the art that the Culture in any known culture medium. Choosing a suitable culture medium is This is within the technical level of the field. The growth promoting substance expected to be used in the present invention is , specifically recognizes and destroys cells that cause suffering to patients, such as cancerous or virus-infected cells. selected for their ability to proliferate lymphocyte subpopulations in vitro. Book In other embodiments of the invention, the cells, in addition to at least one growth promoting substance, Grown in the presence of at least one target antigen. In yet another aspect of the invention isolated lymphoid cells, modified them using genetic engineering methods, and produced them in hollow fiber biomass. Injected into the extra-hollow fiber space of the actor to allow genetically modified subpopulations to proliferate. Culture under the planned conditions.

After inoculation, the hollow fiber bioreactor is opened by applying pressure from the outside with a pump, etc. Perfuse the medium continuously throughout. Glass reservoirs, hollow fiber bioreactors, and and the pumping means are connected by tubing, typically silicone rubber. This piping , to supplement oxygen and whether the medium is buffered by bicarbonate. For example, as a membrane gas exchanger to maintain pH via CO2 transport into the perfusion medium. and operate simultaneously.

In vitro proliferating lymphoid cells grow and divide until they fill most of the extrahollow fiber space. , overlap each other to form a cluster of cells. As cells grow and divide Therefore, the perfusate can be replaced and the EFS can be drained periodically.

The perfusion medium can be changed by changing the reservoir bottle. cell After stable growth, there is no need to include human serum albumin in the perfusion medium. I discovered that.

The EFS is run periodically to collect the supernatant and/or to sample the cells. The fluid can be drained directly. When draining the EFS, any cells that were expelled were also collected and re-inoculated into the hollow fiber bioreactor to complete AIMV or other Cultured in a hollow fiber bioreactor until it contains 1011 cells. . Shake the bioreactor and inject the cells into the side bottle with EFS medium. By doing so, cells can be collected. Furthermore, IL-2 receptor and Other drugs useful for expanding desired lymphocyte cell subpopulations in vitro Contains useful biologically active factors such as growth promoters and is non-diffusible or poorly diffusible. The EFS cell supernatant enriched with biological cell products can be further processed to reveal the biological activity. The active substance can be recovered for purification or partial purification.

Cells are spun and pelleted using a centrifuge or other means known to those skilled in the art. cells, cell pellets, and biologically active components such as IL-2 receptors and growth promoters. It is possible to obtain EFS cell supernatants that are enriched in cells.

After harvesting the cells, by restarting perfusion with the culture medium containing growth-promoting substances. The remaining cells in the bioreactor can then be grown again. cells are the Afterward, it continues to divide and can be recovered. This process can be repeated many times .

After harvesting and pelleting cells, fresh tissue culture is added to generate EFS-conditioned media. Dialysis of EFS cell supernatant against culture medium can be performed. This EFSm culture Cells can also be treated later and stimulate the growth of cells such as TIL cells. It can also be used directly or diluted for use.

In one exemplary procedure using the methods of the invention, from a patient with malignant melanoma The tumor was excised, cut into small pieces, and treated with collagenase 10 mg/ml, deoxyribonucleate. clease 1 mg/m1, and hyaluronidase 2.5 units/ml ( RPMl 1640 tissue culture medium (Biofluids, R.O.P. Kubiru, M.D.). All operations involving cells are performed in a laminar flow hood. Perform using aseptic technique.

After stirring the suspension overnight, it was filtered through a Nitex mesh and I Culture by resuspending in LAK supernatant supplemented with complete AIM-V medium containing L-2. After that, after further growth of the prepared cells, about 50 ml was grown into a hollow fiber bioreactor. inoculate the vector. Autoclave and recirculate the hollow fiber culture system before use. Continuously irrigate, drain and rinse with 1.3 liters of deionized water. Both the FS and perfusion channels are perfused with complete AIM-V medium.

Transfer the inoculated bioreactor to a standard incubator until the Incubation is continued for at least about 10 days to about 30 days. incube glucose and other diffusible nutrients in the EFS at sufficiently high concentrations during the Replace the reservoir containing the perfusion medium for maintenance. If desired, incubate To sample cells or collect EFS cell supernatants during the In addition, the EFS can be drained periodically.

When the desired cell concentration is reached, shake the hollow fiber bioreactor vigorously and remove the EFS. Collect cells by draining. Pellet cells and replace EFS cell supernatant. collected for processing. Recovered cells are characterized by TIL cell morphology and biology. It has certain characteristics.

EFS cell supernatant was added to fresh tissue culture medium to generate EFS conditioned medium. re-dialyzed and added to the newly seeded cells to stimulate growth of the newly seeded cells. It is possible to add. EFS also occurs when cells resume growth after harvesting. , into the hollow fiber bioreactor to stimulate the growth of the cells remaining within it. It can also be added.

The following examples are for illustrative purposes and are not intended to limit the scope of the invention. It's not what I intend.

Example 1 Before use, the eight hollow fiber culture systems were autoclaved at 121°C for 20 minutes; This was followed by perfusion with 1.3 liters of deionized water overnight at 37°C. Keep your reservoir fresh and warm Before replacing with a 1 liter bottle of complete media, clean the perfusion channels and hollow fiber outside of each system. The space was drained and flushed with complete AIMV medium.

All operations were performed in a sterile laminar flow hood.

Tumors were excised from eight patients with metastatic melanoma (listed in column 1 of Table 1 ). Tumors are processed separately from each patient's cells in a laminar flow hood from the operating room. It was transported aseptically. Each patient's tumor tissue was shredded into 1 mm to 2 mm pieces and collated. Genase 10mg/ml, deoxyribonuclease 1mg/m1, and RPM116 containing hyaluronidase 2.5 units/ml (Sigma) 40 tissue culture medium (Biofluids, Rockville, MD) approx. The mixture was suspended in 500ml of water. After stirring each suspension slowly overnight at room temperature, Filter through a Nitex mesh, wash twice, and replenish LAK supernatant. Total AIM-V medium or 10% heat-inactivated human serum, 20% LAK supernatant and I Complete AIM-V and RPM containing L-2 (1000 units/m1) resuspended in either a 50:50 mixture of I.

Each suspension of cells was placed in a 6-well culture plate (Costar Co., Ltd.) at a concentration of approximately 5 x 105 cells/ml. , Cambridge, M.N.). Approximately - weeks later or TIL cell concentration The cells were replated into fresh medium at a concentration of approximately 1-2Xml. After further growth, fine The cells were placed in complete AIM-V medium 0. 5 x 105 cells/in 5 to 1.5 liters It was diluted to a concentration of ml. Then transfer the entire volume to a 1.5 liter polyolefin culture bag. (PL-732 Plastic, Fenwal Laboratories Diaf. in a flat position on a perforated shelf, without stirring. Incubate in a humidified 5% CO2/air incubator for 3-4 days at 37°C. I bet. As cells grow, periodically add a new bag of complete AIM-V medium. It was diluted 1:3 to 4 times underground. Once inoculated into the bag, the cells, after an initial incubation period, It has entered the logarithmic growth phase.

17 to 33 days after incision of the tumor' while the cells in the bag are in logarithmic growth phase. A small amount of cell suspension was centrifuged at 400×g for 10 minutes at room temperature. 10 precipitated cells Complete AI of 0m1 glass side port bottle Resuspend in MV medium. cells from each of eight patients with metastatic melanoma. A hollow fiber culture system was inoculated. Cells that are continuously cultured in bags are treated with the method of the present invention. It was used as a control for comparison with cells cultured according to the method.

Cells obtained from each patient (B, M, S, K, J, H, W and G) were The hollow fiber cartridge and bag were inoculated 1-.

The seeded cells ranged from 0.35 to 4.3 x 108 cells. cells, TI Each was connected to a 100ml glass bottle in which L cells were suspended. was injected into the outer hollow fiber space of the hollow fiber bioreactor through the side port. 20 Gently apply pressure to the bottle with a plastic syringe to make the cell suspension hollow. It was pushed into the extrathread space. The cells are nourished by a diffuse flow from the perfusate. The fiber has settled on or near the fiber. At the same time, low molecular weight metabolites It was released from the vesicle and diluted by perfusate through the fiber.

Two of the eight cultures, S and J (see Table 1), are hollow fiber cultures and control bags. Growth was stopped at . Separate incubators for hollow fiber culture and bag culture The growth arrest was cell-related, as the cells were maintained in culture. It does not seem to be related to the method used. The remaining six cultures are hollow It grew well both in the thread culture system and in bags. Figure 1 shows details obtained from patient W. It represents the cell growth curve.

The system was operated in a humidified 5% CO2/air incubator at 37°C. . During the treatment period, the storage bottle containing the perfusion medium, complete AIM-V, is Replace regularly when the base concentration decreases to a range of 100 to 50 mg/di. did. Other nutrients, as well as glucose, can be added to the bottle containing the medium in a fresh, pre-warmed container. Refill by replacing the bottle with fresh medium. Number of times the bottle was replaced is shown in the 8th column of Table 1. Therefore, the amount of medium used for each culture is Depending on the amount of glucose consumed, 8-27 liters per hollow fiber culture system is within the range of This is approximately a complete AI MV per 1010 collected TILs. This corresponds to 2.4 to 6.6 liters (see bottom column of Table 1). replace storage bottle The time required for this was less than 5 minutes.

Once you start cell culture in a hollow fiber bioreactor, you no longer need human blood in the perfusion medium. We have found that it is not necessary to add clear albumin. Cells are perfused with human blood They grow essentially the same in the absence of clear albumin as in the presence. Ta.

Non-diffusible products that periodically drain the extra-hollow fiber space and are secreted by TIL cells Cells can be harvested to collect enriched culture medium or to assess cell number and functional properties. sampled. The number of times EFS was ejected for each culture is shown in column 9 of Table 1. show. The EFS medium, except for culture G, was replaced on average every 2.2 days. E of culture G FS was exchanged 2 and 0 times within a culture period of 14 and 21 days, respectively.

Slowly drain the fiber external fluid into one of the two loading side-mouth bottles. Then, the space outside the hollow fiber was drained using a laminar flow hood. The medium drained from the EFS was Centrifuge at 00xg for 10 minutes at room temperature and transfer the cell pellet to fresh complete AI M-V medium. and re-seeded into EFS in a hollow fiber bioreactor.

Therefore, any TIL that was washed away was not discarded.

EFS cell supernatants were saved for further processing. Bioreactor effluent, sedimentation, Resuspension and reinoculation took 20-30 minutes.

As the number of cells in the bioreactor increases, the flow rate of perfusate is increased from 40 ml. The speed was increased to 300 m1/min. The direction of perfusion was reversed every 10 minutes. collect cells Inoculation was continued from the 14th to the 32nd (see Table 1) when the animals were ready.

Example 2 After 14 to 32 days, remove all hollow fiber culture systems from the incubator. and placed in a laminar flow hood. Then reinstall the electronic control unit into the pump motor. approximately 40 m to prevent cultured cells from becoming oxygen deficient during the collection procedure. Perfusion continued at a rate of 1/min. Approximately 1/3 of the extrafiber medium is loaded by gravity. Drained into a side-mouth bottle.

The hollow fiber reactor was vigorously shaken to separate the cells from the fibers. . The remaining medium and cells in suspension were drained into a side-neck bottle. Then follow this step Repeated 3 times. This is done by adding fresh water to the side port prior to shaking the bioreactor. This was carried out by putting 35 ml of a complete medium and injecting it into the space outside the hollow fiber. Manual bar A final two times by shaking the cartridge for 1 minute using an iblator. Washing was completed to remove cells that adhered fairly tightly. hollow fiber cartridge Di cells were finally contained in approximately 155-250 m1fi.

On the other hand, an equal number of cells grown in bags will have a gas permeability of at least about 10-20 It is contained in a culture bag or about 15-30 liters of culture medium.

After collection, cells from each bioreactor were centrifuged to form the final pellet. . This is preparation for next use. The entire recovery procedure takes approximately 30 minutes and Approximately 95% of the cells in the ioreactor were recovered. Each hollow fiber bioreactor has 1 ．． 5-5.4 x 1010 cells were produced.

Cell data for each of the eight patients is summarized in Table 1. Collected from each hollow fiber culture system The number of cells obtained is shown in the fifth column.

Example 3 1.5X101O TILs from patient G on day 30, i.e. bioreactor The cells were collected from the bioreactor on the 15th day after inoculation. Complete AIM-V perfusion of the bioreactor was resumed. The remaining cells in the EFS divide and multiply. Continued to grow and produced an additional 1.5 x 1010 cells on day 21 (Table 1 and Figure 2 reference). Restart perfusion and allow remaining cells to grow again for a third harvest. On day 73, 2.1 x 1010 cells were collected.

After the final harvest, all Ceramax hollow fiber culture systems were heated to 121°C. It was autoclaved for 20 minutes. The system is then flushed with deionized water and the silicone The rubber tubing and hollow fiber bioreactor were discarded. before reuse on different patients. Cleaning, reassembling and repressurizing new piping and bioreactors at appropriate times. Sterilization was performed.

Example 4 In vitro analysis of sampled cell suspensions collected from bioreactors. The functional properties of the suspension were compared with the corresponding suspension collected from the bag and sampled. compared.

Add trypan blue/normal to a final concentration of 0.04% gm/di. Periodically and after collection by diluting in normal saline (Sigma) Cell viability assays were performed. Viability observed under microscope inspection Evaluation was made by the exclusion of the dye. Cell viability shown in Table 1 varies between patients. It became. The average survival rate of cells grown in the hollow fiber culture system was over 90%. .

During the culture period, aliquot a portion of the perfusion to determine glucose and lactose concentrations. (Yellow Springs Instrument Co., Yellow Springs) ngs, oh). This measures the consumption rate and production rate, respectively. be able to. Referring to FIGS. 1 and 2, glucose by TIL of different patients The gas consumption rate varies between 0.45 and 2.2 g/1010 TI L/24 hours. (Table 1.7 column).

The production rate of lactic acid was almost the same as the consumption rate of glucose. This rate increases logarithmically. and doubles every 1.5 to 3.6 days. These doubling times are grown in bags This was commensurate with the cell doubling time of 1,5 to 3.2 days.

Cell cytotoxicity to autologous tumor cells, allogeneic tumor cells, and high NK sensitivity562 against targets consisting of erythroleukemia cell lines and NK-resistant Daudi B-cell lymphoma lines. Evaluated by tested chromium release assays (e.g. Topalian el al.

(1987) J, 1mmuno1. (See Meth, 102:127) . The results of the cytotoxicity studies are listed in Table 2. This is a simultaneous method of bag and hollow fiber culture. Cell lysis ability measurement results of three TIL dilutions obtained from aliquots collected in It shows.

TIL cells collected from patients M, H, W and G at different times during the proliferation period. 562 cells, Daudi cells, autologous tumor cells, and allogeneic cells. Percentage of specific lysis of atypical tumor cells by chromium release assay It was measured. For all cell lines, as well as autologous and allogeneic tumor cells. The percentage of specific lysis of LAK cells with lytic ability was also determined.

Since the K562 cell line is highly sensitive to NK, in vitro expanded lymphoid cells obtained from each patient Useful for evaluating NK cell activity of cells. NKHI+ has non-specific damaging activity. Responsible. The NK-resistant but LKA-sensitive Daudi cell line was given The relative amount of LKA activity present in the sample is shown.

Approximately 108 target cells were cultured with 400 μCi of N,M,N, in 0.7 ml. Time-label, wash 3 times, incubate for 30 minutes at 37°C, and use. Washed twice before.

Serial dilutions of sampled effector cells were combined with 5 x 10 target cells. 96-well round bottom microtiter plate (Costar) Effector:target cells were mixed at 80:1.20:1 in a total of 150 μl of culture medium. , and 5:1 in three ratios and incubated for 4 hours. Then the supernatant with the Scatronautitatec φ system (Scatron, Lille, Norway) Gamma Counter (LKB Instruments, Gaitersburg, M/D) was counted. tumor target cells in the absence of effector cells released by naturally occurring target lysis by incubation. The amount of radioactivity was measured. By incubating the target with 2% SDS, The maximum amount of released radioactivity indicating maximum cell lysis was determined.

The percentage of specific lysis is therefore (experimental release - spontaneous release) Equals 100 maximum release - spontaneous release.

Although the scope of the experiment is somewhat limited, from the data shown in Table 2, the hollow fiber bioreactor The relative activities of different cell types present in TIL cell preparations grown in the patient It appears to vary between individuals and even within a patient over time.

For example, let us refer to the data set forth in Table 2 obtained for W-TIL.

Here, the first set was measured 7 days after inoculating the hollow fiber bioreactor, and the second The set was measured on the 28th day. However, one patient whose samples were taken on the same day Compare data for each sample obtained from bag or IF for cells of Certain differences between cells cultured in HF and bags are then revealed. . NK and LAK cell activities were higher in cells grown in HF than in cells grown in bags. The cells appear to be lower. Furthermore, these differences may be associated with this in TIL activity. There appears to be no correlation with the corresponding decrease. Therefore, by the method of the present invention Expanded cells appear to have a lower percentage of TIL-unrelated activities. .

Also representation of cells sampled and recovered from bags and hollow fiber reactors. We also compared the types. The results of these experiments for patients K and G are shown in Table 3.

The sampled or collected cells are placed in cold staining medium (phenol red). Contains 5% heat-inactivated fetal bovine serum and 0.02% sodium azide. 1 x 106 to 1 x 107 cells/ml. resuspended in medium at a concentration of Fluorescein against undiluted human mononuclear cell antigen Conjugated monoclonal antibody (Vectonodekinson, Mountain View, C. Add Ni) at a concentration of 5% (V/V) until the cell suspension reaches 100 μlfi. did.

Using monoclonal antibodies, we can identify the presence on the cell surface and functionally characterize the cells. The surface antigens were confirmed. The antibodies tested were anti-Le, which reacts against T cells; u-4, anti-Leu-3as T cell disorder responding to T helper/triggered cells Anti-Leu-2bx E rosette receptor-containing cells that respond to harmful/inhibitory cells. anti-Leu'-5b, which reacts against cells, NK and some T cells; corresponding anti-Leu-7, anti-Leu that reacts against NK cells and neutrophils; 11a Anti-Leu-14 and anti-Leu-16 reactive against SB cells, Anti-antibiotics that react against activated T cells, B cells, and monocytes/macrophages. HLA-DR, anti-I L-2 receptor that reacts with activated T cells, and and anti-Leu-M3 that reacts against monocytes/macrophages. as a control The antibodies used were mouse T cells and phycoerthrogin. rosin). 30-60 minutes at 4℃ After inter-staining, cells were washed with staining medium, fixed with 1% formaldehyde, Washed again and resuspended in staining medium. Cells labeled in this way were incubated at 4°C for 1~ Stored for 7 days. Consort 40 computer (Bect Nordkinson) Fluorescence analysis using an interface FACS440 microfluorometer carried out.

The results of these experiments demonstrate the surface antigen profile of cells grown using the method of the invention. and the surface antigen profiles of the cells in the bag are virtually identical. ing. Surface antigens present on the surface of cells grown in bags can also be used in the method of the invention. present on the surface of cells grown in the bag and substantially present on the cells grown in the bag. Absent surface antigens are also substantially absent from the surfaces of cells grown by the method of the present invention. do not have.

TIL IIF Culture Inoculated cells Collection cells Survival rate Gluco 5 Riza # back Total medium Medium amount B 33 21 0.5 4.5 85 3.6 12 10 11.3 2.5M 31 32 0.35 4.1 97 3.5 25 14 27 6.6S 1.3 - K 58 29 10.0 26m1 1.9 12 10 13J 0.4 − [124261,82,8911,7912103,6W 24 22 4.3 5.4 93 2.4 12 7 13 2.4G 17 14 1.0 1 ．． 5 87 2.9 7 2 1 + 5.0G 31 21 Residue 1.5 8 9 3.3 8 0 8.8 5.8*HF = Hollow fiber culture system.

Patients whose tumors were removed to produce TrL==TIL.

Cells from patients S and J did not grow in either bags or HF.

Days of HF inoculation = Days after dissecting the tumor from the patient and inoculating the cells into the HF device.

Survival rate = percentage alive at time of collection.

Glucose gm/d = Glucose consumption of collected cells # Replacement of reservoir bottle Number of times = number of times the medium in the reservoir was changed during the HF culture period.

: Number of times EFS was replaced = number of times the outer fiber space was replaced during the HF culture period.

Table 2 Efnik 9- K562 Daudi autologous, allogeneic: am 80:l 20:1 5:1 80:1 20:1 5:l 80:1 20:1 5:1 80:1 20+1 sat effector cell Mine J44.412.96.928.49.15.7M-Setting (Il [F) 6.6 5.3. 8 2.5 1.1 4.1-TIL (Bag) 58.733. 011519. In, 53.7 part (HF) 21.611.27.44.85 ．． 04.5LAK65.223.07.343.614.18.417.28. 94.34.61.13, 6H-Tn, CJ3ag:) 7.4 5.8. 8 2.5 5.6 6.6 50.2 57.1 33.7 B, 6. 1 7 ．． 3H-TIL (EIF) 2.3. 9. 7. 8 2.8 2.8 49 ．． 9 42.9 39.6. 8 5.0. 6-TIL(HF)4.21. 3.81.82.13.13.64.9.1Lλ to 81.044.018.03 8.818.66.05.1.92.09.16.6.9Y-TIL (Bag) 30.524, 522.764, 053.229.230, 614.93.24 ．． 86.84.5W-TI13) 22,511.212.555.145.01 9.819.411.94.42.04.15.8 LA to 8[i, 1175.6 27.469.845.817.234.313. fli4,126.011. 74.5W-Tn, (Bag) 15.06.9.972.253.121,71 5.47.92.85.512.72.5W-Tn, (JIF)8.62.5, 355.941.317.011.502.03.95.45.0LAK74. 356.149.959.745,940,069.941,318,137, 143,117.7G-TIL(Bag)13.58.55,221.34.9 2,651,132.022.33.92.92.5G-TIL(U)11.1 10.16.13.99.67.650,440,438.23.43.23. 6th day 4 94.1 94.7 85.3 91.4 cases 5 Steam autoclave the hollow fiber culture system at 121°C for 20 minutes as in Example 1. Processed with. It was then perfused with 1.3 liters of deionized water at 376C overnight. So After draining the perfusion channels and the space outside the hollow fibers of each system, the storage container was newly heated. Flush with complete AIM-V medium before replacing with complete medium in the sotol container . All operations were performed in a sterile laminar flow hood.

Tumors were excised from cancer patients and transported aseptically from the operating room to a laminar flow hood. Here, the tumor Cells from the tumor were cut into 1 mm to 2 mm strips, approximately 200 to 500 ml. RPMI 1640 tissue culture medium (Biofluids, Rockville , MD).

This tissue culture medium contains 10 mg collagenase per ml, 1 equivalent per ml. 1 mg of deoxyribonuclease per cubic meter and 2.5 units of hydrogen per 1 m Contains aluronidase (S i gma). The suspension is stirred gently at room temperature overnight. Stirred. Next, the suspension was treated with sterilized Nitex mesh. filtered through, washed twice and added LAK supernatant as described in Example 1. Resuspended in AIM-V medium.

A portion of the suspension, i.e. at least 50 ml, is sufficient to inactivate the tumor cells. irradiation by time, X-rays or other suitable method. Irradiated suspensions are known to those skilled in the art. stored in liquid nitrogen in a non-freezing medium. Immediately before use, this suspension Warm to ℃.

A portion of the remaining suspended cells was added to 6 cells at a concentration of approximately 5 x 105 cells/ml. culture plate with wells (Coslar Corp.).

sown in Cambridge, MA). Approximately - weeks later or TIL cells When the cell concentration reaches approximately 1-2X106 cells/ml, the cells are divided into approximately 5x105 cells/ml. /ml in fresh medium and incubate until at least 107 to 108 cells. fed. Cells were pelleted and resuspended in a suspension containing irradiated tumor cells. . The volume was adjusted to approximately 100 ml with complete AIM V medium. This mixture was prepared in Example 1 The cells were planted in EFS in a hollow fiber culture system as described in . The cells are on the hollow fiber surface. settled on or near a surface.

The culture system was operated as described in Example 1. First, the perfusion medium was completely AIM- V, which means that the concentration of glucose is approximately 1 to 1.5 grams per liter. When the ram was reduced, it was replaced periodically. Cell culture in hollow fiber bioreactors Once established, it is no longer necessary to add human serum albumin to the perfusion medium.

The perfusate flow rate was increased by 4 to increase the number of cells in the bioreactor over time. Increase from 0 to 300 m1/min.

The direction of perfusion is reversed every 10 minutes. Continue culturing for 14 to 32 days, during this period In the meantime, the cells were taken as described in Example 1, and the hollow fiber culture system was placed in the hollow fiber culture system for 12 hours as in Example 1. Steam autoclaved at 1°C for 20 minutes. Next, 1.3 liters of deionized water overnight at 37°C. Irrigation path and space outside the hollow fibers of each system before draining and replacing the storage container with a freshly warmed 1 liter container of complete medium. , flushed with complete AIM-V medium. All operations are performed in a sterile laminar flow hood I went inside.

Tumors were excised from cancer patients and transported aseptically from the operating room to a laminar flow hood. Here, the tumor Cells from the tumor are cut into 1 mm to 2 mrn pieces, approximately 200 to 500 m1. RPMI 1640 tissue culture medium (Bio[1uids, Rockville e, suspended in MDl.

The tissue culture medium contained 10 mg collagenase per ml; 1 mg of deoxyribonuclease and 2.5 units of hyaluronan per ml Dase (S i gma) is included. The suspension was stirred gently at room temperature overnight. . Then filter through a sterile Nitex mesh and wash twice. and resuspended in complete AIM-V medium supplemented with LAK supernatant, as described in Example 1. It got cloudy.

A portion of the suspension was removed, ie at least about 50 ml.

The suspension was mixed with microparticle carrier beads. The above cells are placed on these microparticle carrier beads. Combine to form a slurry. Add enough of this slurry to make the tumor cells inactive. irradiated with X-rays for a period of time. This slurry is then introduced into the bioreactor and The particle carrier beads were anchored at or near the hollow fiber surface.

Alternatively, a portion of the suspension of tumor cells may be added until the tumor cells are no longer proliferating. It was irradiated with X-rays for a sufficient period of time. This suspension of tumor cells was then placed in an HF bioreactor. The microparticle carrier beads are introduced into the EFS of the hollow fiber, and the microparticle carrier beads are fixed on the hollow fiber surface or its vicinity. It was to so. Introducing non-toxic cross-linking reagents into EFS to prevent tumor cells from joining the hollow fibers Fixed in reverse. After cross-linking, the EFS was drained and replaced with complete AI M-V medium. perfused.

The remainder of the suspended cells was added to 6 wells at a concentration of approximately 5 x 10 cells/ml. A certain culture plate (Co'5tar Corp.

Camb r i-dge, MA). After about - weeks or '', TIL When the cell concentration of the cells reached approximately 1-2X10" cells/ml, the cells were 05 cells/ml in fresh medium to yield at least 107 to 108 cells. The cells were cultured until Cells were pelleted and resuspended in suspension containing irradiated tumor cells. Made it muddy. The volume was adjusted to approximately 100 ml with complete AIM V medium. this mixture were inoculated into EFS in a hollow fiber culture system as described in Example 1. cells are inside It settled on the empty fiber surface or its vicinity.

The culture system was operated as described in Example 1. First, the perfusion medium was completely AIM- V, which means that the concentration of glucose is approximately 1 to 1.5 grams per liter. When reduced to ram, it is replaced periodically. Once cell culture was established, hollow fiber bioreactors Once started in the perfusion medium, it is no longer necessary to add human serum albumin to the perfusion medium. stomach.

The perfusate flow rate was increased by 4 to increase the number of cells in the bioreactor over time. It was increased from 0 to 300 m1/min.

The direction of perfusion was reversed every 10 minutes. Continue culturing for 14 to 32 days, during this period In the meantime, cells were removed as described in Example 1.

Steam autoclave the hollow fiber culture system at 121°C for 20 minutes as in Example 1. Processed with. The cells were then perfused with 1.3 sotol of deionized water overnight at 37°C.

The irrigation channels and the space outside the hollow fibers of each system were drained, and the storage containers were newly heated. Flush with complete AIM-V medium before replacing with complete medium in the bottle. . All operations were performed in a sterile laminar flow hood.

Tumors were removed from cancer patients and transported aseptically from the operating room using laminar flow. Here, the tumor Cells from the tumor were cut into 1 mm x 2 mm strips, approximately 200 to 5 00ml RPM11640 tissue culture medium (Biofluids, Rackv ille, MD).

This tissue culture medium contained 10 mg collagenase per ml, 1 mg of deoxyribonuclease per cubic meter and 2.5 units of hydrogen per 1 m Includes aluronidase (Sigma). The suspension was stirred gently at room temperature overnight. did. Next, filter through a sterilized Nitex filter, Complete Al11-V medium, washed twice and supplemented with LAK supernatant, as described in Example 1. resuspended in

This cell suspension was placed in a 6-well culture plate at a concentration of approximately 5X105 cells/ml. Rate (Costar Corp, Cambri-dB.

MA). After about -10 weeks or from a cell concentration of TIL cells of 108 cells The cells were cultured until the following occurred. Pellet the cells and add 1000 units/ml IL- resuspended in complete AIMV medium containing 4. This mixture was prepared as described in Example 1. The cells were planted in EFS, a hollow fiber culture system. Cells are located on or near the hollow fiber surface. It took root in

The culture system was operated as described in Example 1. First, the perfusion medium was completely AIM- V or complete AIM-V, which also contained 1000 units/ml of IL-4. . The perfusion medium reduces its glucose concentration to approximately 1 to 1.5 grams per liter. I replaced it periodically after a while. Once the cell culture is in the hollow fiber bioreactor, Once started, it is no longer necessary to add human serum albumin to the perfusion medium.

The perfusate flow rate was increased to 4 so that the number of cells in the bioreactor increased throughout the period. It was increased from 0 to 300 m1/min.

The direction of perfusion was reversed every 10 minutes. Continue culturing for 14 to 32 days, during this period In between, remove the cells as described in Example 1 from the hollow fiber bioreactor as in Example 1. After removing the cultured cells and EFS medium, the EFS and cells were centrifuged at 200Xg. The cells were pelleted using a heart separator. EFS cell culture supernatant with fresh complete AI Dialyzed against MV medium for 24 hours at 4°C, called EFS conditioned medium. produced.

104 cells were seeded into flasks and incubated with complete AIMV at 1:10 or in 5 ml of EFS conditioned medium diluted 1:100 or in dialyzed complete Cells were grown to double in number in 5 ml of total AIM V. EFS training The medium was found to stimulate cell proliferation. The data are shown in Table 4.

Table 4 Stimulation of proliferation of TILs by EFS cell culture supernatants Dilution of EFS 1:100 1:10 Growth in EFS conditioned medium 20 12.7 Cell number XIO' 16.8 12. 7 Growth in dialyzed medium 9.210.0 cell count x lO' 9. 0 13. 2 Since modifications will be apparent to those skilled in the art, the invention is covered only by the appended claims. should be limited to.

Culture period (days) FIG. 2 Submission of translation of written amendment (Article 184-8 of the Patent Law) March 16, 1992

Claims (40)

[Claims] 1. A method for producing in vitro proliferating lymphoid cells that can be used in adoptive immunotherapy. So, (a) Hollow fiber of hollow fiber bioreactor, which is one component of hollow fiber culture system inoculating the external space with a suspension of lymphoid cells; (b) a growth promoting substance that specifically proliferates a therapeutically useful subpopulation of said lymphoid cells; perfusing the bioreactor with a tissue culture medium containing an effective amount of at least one of , wherein the effective amount is an amount sufficient to effect the specific growth and the tissue culture medium is maintains cell division and growth of the subpopulation, and the therapeutic use is adoptive immunotherapy. and (c) the cells in the bioreactor are treated in the presence of the growth promoting substance. Incubate the cells for a sufficient period of time to obtain a therapeutically effective number of the in vitro proliferating lymphoid cells. where the therapeutically effective number is low when the cells are used for adoptive immunotherapy. At least the number of cells is sufficient to achieve a therapeutic effect. 2. The growth promoting substance is selected from the group consisting of mitogens and cytokines. 2. The method according to claim 1, wherein the at least one substance is at least one substance. 3. The cytokine may be interleukin-1, interleukin-2, or Cytokine selected from the group consisting of tarleukin-4 and interleukin-6 3. The method according to claim 2, wherein the method is at least one type of in. 4. A claim further comprising recovering the contents of the extra-hollow fiber space of the bioreactor. 1. The method according to item 1, wherein the contents are grown in the in vitro proliferating lymphoid cells and A method involving cell supernatants and extrahollow fiber spaces. 5. A claim further comprising recovering the contents of the extra-hollow fiber space of the bioreactor. 3. The method according to item 3, wherein the contents are grown in the in vitro proliferating lymphoid cells and A method involving cell supernatants and extrahollow fiber spaces. 6. Resetting the perfusion of the bioreactor and resetting the bioreactor after the first withdrawal. Any residual cells remaining in the extra-hollow fiber space of the reactor are removed by adding a therapeutically effective amount of said cells. 5. The method according to claim 4, further comprising culturing for a sufficient time to obtain and the perfusion and recovery are reset at least once. 7. resetting the perfusion of the bioreactor and the device after the first withdrawal. to remove residual cells remaining in the extra-hollow fiber space in a manner sufficient to obtain a therapeutically effective amount of said cells. 6. The method of claim 5, further comprising culturing for a period of time, and how collection is reconfigured at least once. 8. Claim 1, wherein said medium also contains an effective amount of at least one target antigen. 2. The method according to paragraph 1, wherein the effective amount is at least a small amount that specifically recognizes the antigen. Sufficient to select for proliferation of at least one proliferating lymphoid cell subpopulation in vitro Law. 9. At least one of the target antigens is selected from the group consisting of tumor-specific antigens. The method described in item 8 of the scope of the request. 10. The target antigen is present only in the medium within the extra-hollow fiber space of the bioreactor. 9. The method according to claim 8. 11. At least one of the target antigens is selected from the group consisting of tumor-specific antigens. The method according to claim 10. 12. Cells are pelleted and removed from the contents of the hollow fiber outer space, and the cells are pelleted and removed from the contents of the hollow fiber outer space. 5. The method of claim 4, further comprising producing a cell supernatant. 13. The hollow fiber extraspace cell supernatant was dialyzed against tissue culture medium to remove the hollow fiber extraspace. 13. The method of claim 12, further comprising producing a conditioned medium. 14. The genome of the lymphoid cell is expressed when the cell is used for adoptive immunotherapy. modified by inserting cloned DNA encoding a protein that The method according to claim 1. 15. The cloned DNA contains a traceable marker protein, a therapeutically effective Consists of proteins and proteins that can cause drug resistance and susceptibility Claim 14, which encodes at least one protein selected from the group How to put it on. 16. An improved method for growing proliferating lymphoid cells in vitro, comprising: Tumor-infiltrating lymphocytes; (a) cells derived from resected tumor tissue suspended in cell tissue culture medium; Make it muddy, (b) the presence of at least one cytokine capable of promoting proliferation of tumor-infiltrating lymphocytes; culturing the suspension in the presence of an effective amount, wherein the effective amount The amount is sufficient to cause proliferation of tumor-infiltrating lymphocytes, (c) said culture suspension of tumor-infiltrating lymphocytes in one component of a hollow fiber culture system; Inoculate the space outside the hollow fiber of a certain hollow fiber reactor, (d) the bioreactor is capable of promoting proliferation of tumor-infiltrating lymphocytes; perfused with tissue culture medium containing an effective amount of one cytokine; the amount is sufficient to effect the specific growth, and the tissue culture medium is sufficient to cause the tumor infiltration. maintain cell division and growth of lymphocytes; and (e) inhibit said tumor-infiltrating lymphocytes. Within the bioreactor, in the presence of the tissue culture medium, a therapeutically effective number of culture for a sufficient period of time to obtain the tumor-infiltrating lymphocytes, where the therapeutically effective number is at least enough to achieve a therapeutic effect when the cells are used in adoptive immunotherapy. the number of said cells. 17. The cytokine may include interleukin-1, interleukin-2, and interleukin-2. Site selected from the group consisting of interleukin-4 and interleukin-6 17. The method according to claim 16, wherein at least one type of caine is used. 18. The claim further includes recovering the contents of the extra-hollow fiber space of the bioreactor. Claim 16. The method according to claim 16, wherein the content comprises the tumor-infiltrating lymphocytes and the tumor-infiltrating lymphocytes. A method involving hollow fiber extraspace supernatant. 19. Resetting the perfusion of the bioreactor and resetting the bath after the first withdrawal. The residual tumor-infiltrating lymphocytes remaining in the space outside the hollow fibers of the bioreactor are removed from the tumor-infiltrating lymphocytes. of the claim further comprising culturing the lymphocytes for a sufficient period of time to obtain a therapeutically effective amount of the lymphocytes. The method of scope 18, wherein the perfusion and withdrawal are reset at least once. How to do it. 20. The medium also contains an effective amount of at least one tumor-specific target antigen. The method according to item 18, wherein the effective amount is at least specific for the antigen. sufficient to select for proliferation of at least one tumor-infiltrating lymphocyte subpopulation that recognizes How to be. 21. The target antigen is present only in the medium within the extra-hollow fiber space of the bioreactor. 21. The method of claim 20 introduced. 22. Cells are pelleted and removed from the contents of the hollow fiber outer space, and the cells are pelleted and removed from the contents of the hollow fiber outer space. 19. The method of claim 18, further comprising producing a cell supernatant. 23. The hollow fiber extraspace cell supernatant was dialyzed against tissue culture medium to remove the hollow fiber extraspace. 23. The method of claim 22, further comprising producing a conditioned medium. 24. The genome of the tumor-infiltrating lymphocytes indicates that the lymphocytes were used for adoptive immunotherapy. By inserting cloned DNA encoding the protein to be expressed in the 2. The method of claim 1. 25. The cloned DNA contains a traceable marker protein, a therapeutically effective Consists of proteins and proteins that can cause drug resistance and susceptibility Claim 24, which encodes at least one protein selected from the group How to put it on. 26. (a) a hollow shell suitable for the growth of mammalian cells; (b) contained within the shell; a plurality of hollow fibers, the hollow fibers being semi-permeable and having a and a hollow fiber suitable for the growth of mammalian cells in the vicinity; (c) a surface of the hollow fiber; an increase in at least one subpopulation of lymphoid cells on or near the lymphoid cells; in the presence of an effective amount of at least one growth promoting substance that specifically promotes growth. The subpopulation is cultured and the subpopulation contains lymphoid cells, which can be used for adoptive immunotherapy. call (d) a tissue culture medium surrounding the hollow fibers within the shell, the tissue culture medium surrounding the hollow fibers; at least one growth promoter that specifically proliferates a therapeutically useful subpopulation of parathyroid cells. containing an effective amount of a substance, said effective amount being an amount sufficient to effect said specific proliferation; Tissue culture media maintains cell division and growth of the subpopulation and supports the therapeutic use. A hollow fiber bioreactor equipped with a culture medium, which is an adoptive immunizer. 27. The growth promoting substance is selected from the group consisting of mitogens and cytokines. 27. The bioreactor according to claim 26, wherein the bioreactor is at least one of the following substances: . 28. The cytokine may include interleukin-1, interleukin-2, and interleukin-2. Site selected from the group consisting of interleukin-4 and interleukin-6 28. The bioreactor according to claim 27, wherein the bioreactor is at least one type of caine. 29. 27. The biorea of claim 26, wherein the lymphoid cells are tumor-infiltrating lymphocytes. ctor. 30. In vitro proliferated lymphoid cells produced by the method according to claim 1. 31. In vitro proliferated lymphoid cells produced by the method according to claim 6. 32. In vitro proliferated lymphoid cells produced by the method according to claim 14 . 33. A tumor-infiltrating lymphocyte produced by the method according to claim 16. 34. A tumor-infiltrating lymphocyte produced by the method according to claim 19. 35. A tumor-infiltrating lymphocyte produced by the method according to claim 24. 36. A hollow fiber extracellular space supracellular groove produced by the method according to claim 12. 37. A hollow fiber extraspace-conditioned medium produced by the method according to claim 13. 38. A hollow fiber extraspace cell supernatant produced by the method according to claim 22. 39. A hollow fiber extraspace-conditioned medium produced by the method according to claim 23. 40. An improved method for producing in vitro proliferating lymphoid cells comprising an effective amount of hollow culturing the cells in the presence of an extrathalamic space-conditioned medium, the amount of which Stimulating the growth rate of proliferating cells so that they grow less than they would in their absence. How to increase it by at least about 50%.